Solution 1 (see below)...................4.0 ml
Solution 2 (see below)...................4.0 ml
NaHCO3 ...................................0.8 g
Yeast extract............................4.0 g
Tryptone.................................4.0 g
AQDS*....................................3.3 g
Wolfe's Vitamin Solution (see below).....0.4 ml
Wolfe's Mineral Solution (see below).....0.48 ml
Distilled water........................392.0 ml
*9,10-Anthra-quinone-2,6-disulfonic acid Combine above ingredients, except for yeast extract and tryptone.
Dissolve and bring to boil under 80% N2, 10% CO2, 10% H2.
Remove from heat and allow to cool. Add the yeast extract and tryptone. Adjust pH to 7.2 +/- 0.2.
Dispense under gas into aluminum seal tubes, 10.0 ml per tube. Autoclave at 121C for one hour.
Solution 1:
NH4Cl ........................1.65 g
KCl..........................1.65 g
MgCl2 . 6H2O .................1.65 g
CaCl2 ........................1.65 g
Distilled water.............50.0 ml
Solution 2:
KH2PO4 .......................1.65 g
Distilled water.............50.0 ml
Wolfe's Vitamin Solution:
Available from ATCC as a sterile ready-to-use liquid (Vitamin Supplement, catalog no. MD-VS).
Biotin.......................2.0 mg
Folic acid...................2.0 mg
Pyridoxine hydrochloride....10.0 mg
Thiamine . HCl...............5.0 mg
Riboflavin...................5.0 mg
Nicotinic acid...............5.0 mg
Calcium D-(+)-pantothenate...5.0 mg
Vitamin B12..................0.1 mg
p-Aminobenzoic acid..........5.0 mg
Thioctic acid................5.0 mg
Distilled water..............1.0 L